Abstract

Breast cancer is a heterogeneous disease, exhibiting a wide range of molecular aberrations and clinical outcomes. Here we employed paired-end transcriptome sequencing to explore the landscape of gene fusions in a panel of breast cancer cell lines and tissues. We observed that individual breast cancers harbor an array of expressed gene fusions. We identified two classes of recurrent gene rearrangements involving microtubule associated serine-threonine kinase (MAST) and Notch family genes. Both MAST and Notch family gene fusions exerted significant phenotypic effects in breast epithelial cells. Breast cancer lines harboring Notch gene rearrangements are uniquely sensitive to inhibition of Notch signaling, and over-expression of MAST1 or MAST2 gene fusions had a proliferative effect both in vitro and in vivo. These findings illustrate that recurrent gene rearrangements play significant roles in subsets of carcinomas and suggest that transcriptome sequencing may serve to identify patients with rare, actionable gene fusions.

Recurrent gene fusions and translocations have long been associated with hematologic malignancies and rare soft tissue tumors as driving genetic lesions 1–3. Over the last few years, it is becoming apparent that these genetic rearrangements are also found in common solid tumors including a large subset of prostate cancers 4,5 and smaller subsets of lung cancer, among others 6. Secretory breast cancer, a rare subtype of breast cancer, is characterized by recurrent gene fusions of ETV6-NTRK37. While a number of breast cancer genomes have been sequenced 8,9, and complex somatic rearrangements observed 10, “driving” recurrent gene fusions have thus far not been identified.

We employed paired-end transcriptome sequencing on a panel of 89 breast cancer cell lines and tumors (Fig. S1), and applied our previously developed chimera discovery pipeline 11,12. This represented a spectrum of breast carcinoma, including 42 estrogen receptor (ER) positive, 21 ERBB2 positive, and 27 triple negative (ER−/PR−/ERBB2−) samples (Table S1). Fusion transcript discovery led to the identification of 384 expressed gene fusions, an average of nearly five per breast cancer sample, with a slightly higher number of gene fusions in the cell lines compared to primary tumors (Fig. S1b and Table S2). Remarkably, only SEC16A-NOTCH1 was found to be recurrent in our compendium, even as several fusion genes did appear in combination with different fusion partners. Overall, 24 genes were found to be recurrent fusion partners, highlighted in Table S2. In order to focus on potentially tumorigenic ‘driver’ fusions, we prioritized the gene fusions based on the known cancer-associated functions of component genes. While there were many singleton fusions in our compendium that met these criteria, we identified 5 cases of fusions of MAST family kinases and 8 cases of fusions of Notch family genes (Fig. 1 and Fig. S2).

MAST kinase family genes are characterized by the presence of a serine/threonine kinase domain, a 3’ MAST domain with some similarity to kinase domains, and a PDZ domain 13. Little is known about the biological role of MAST kinases and somatic alterations have not been described in cancer. Initially, three independent cases of MAST gene fusions were identified by transcriptome analyses-ARID1A-MAST2, ZNF700-MAST1, and NFIX-MAST1 (Fig. 1a). We devised a targeted sequencing approach to screen additional samples for MAST gene fusions. A transcriptome library of 74 pooled breast carcinoma RNAs was generated and captured with baits encompassing MAST1 and MAST2. After sequencing, two new MAST gene fusions were discovered, TADA2A-MAST1 and GPBP1L1-MAST2 (Fig. 1a). The samples harboring MAST gene fusions are distinct from those with Notch family gene fusions (Fig. 1b), discussed later.

The fusions were confirmed by fusion-specific PCR (Fig. 2a). All five MAST fusions encoded contiguous open reading frames, some retaining the canonical serine/threonine kinase domain and all retaining the PDZ domain and the 3’ kinase-like domain (Fig. 2b). Thus overall, we have discovered five novel gene fusions encoding MAST1 and MAST2 in a cohort of a little over 100 breast cancer samples and more than 40 cell lines, suggesting that the novel serine/threonine kinase family gene fusions represent a subset of 3–5% of breast cancers.

The ZNF700-MAST1 fusion transcript encodes a truncated MAST1 protein that retains the 3’ kinase-like and PDZ domains. We cloned the open reading frame of the ZNF700-MAST1 fusion to test phenotypic effects and used a full-length MAST2 expression construct to mimic the function of ARID1A-MAST2 over-expression. To assess the potential oncogenic functions of MAST genes, we ectopically over-expressed epitope tagged- truncated MAST1 and full length MAST2 in the benign breast cell line, TERT-HME1 (Fig. S3a–h). We next cloned and expressed all five MAST1/MAST2 fusions. Consistent with the earlier observations, TERT-HME1 cells overexpressing the five MAST fusions (Fig. 2c) also displayed higher rates of cell proliferation (Fig. 2e). Overall, these results suggest that ectopic expression of the MAST fusions impart growth and proliferative advantage in benign breast epithelial cells.

As the endogenous ARID1A-MAST2 fusion is present in the breast cancer cell line MDA-MB-468, we used multiple independent MAST2 or ARID1A-MAST2 fusion-specific siRNAs to achieve a marked knockdown of the ARID1A-MAST2 fusion protein (Fig. S3i–s). Knockdown of MAST2 showed significant growth inhibitory effects in MDA-MB-468 cells but not in the fusion negative line BT-483, or benign breast cells, TERT-HME1 (Fig. 2d). To further characterize the effects of the ARID1A-MAST2 fusion in MDA-MB-468 cells, we used shRNA targeting MAST2, which displayed efficient knockdown of ARID1A-MAST2 fusion transcript and protein (Fig. S3k–l). MDA-MB-468 cells treated with MAST2 shRNA exhibited a dramatic reduction in growth as demonstrated in a colony formation assay (Fig. 2f), as well as showed increased apoptosis with S-phase arrest (Fig. S3m–n). In the mouse xenograft model, MDA-MB-468 cells transiently transfected with MAST2-shRNA failed to establish palpable tumors over a time course of 4 weeks (Fig. 2g). The knockdown studies show that the ARID1A-MAST2 fusion is a critical driver fusion in MDA-MB-468 cells.

In addition to MAST fusions, a total of 8 rearrangements involving either NOTCH1 or NOTCH2 were discovered (Fig. 1b and Fig. S2). All were found in ER negative breast carcinomas (p = 0.008), and all but one in triple negative breast carcinomas. We focused on one ER negative tumor and three ER negative breast cancer cell lines with 3’ NOTCH1 or NOTCH2 fusion transcripts for functional studies. The Notch fusion transcripts are abundantly expressed and are specific to samples harboring DNA rearrangements (Fig. 3a). All fusion transcripts retain the exons encoding the Notch intracellular domain (NICD), responsible for inducing the transcriptional program following Notch activation (Fig. 3b). The DNA breakpoints associated with Notch fusions were characterized by mate-pair genomic library sequencing, or long-range genomic PCR (Fig. S4a,b).

Identification and characterization of Notch gene aberrations in breast carcinomas

The predicted open reading frames for the NOTCH1 and NOTCH2 fusion transcripts fall into two classes (Fig. 3b). For both the SEC16A-NOTCH1 fusions and the intragenic NOTCH1 fusion in HCC1599, the predicted ORFs initiate after the S2 cleavage site, but before the S3 γ-secretase cleavage site, similar to the TCRB-NOTCH1 fusion in the T cell adult lymphocytic leukemia line CUTLL114. In contrast, the SEC22B-NOTCH2 fusion ORF is predicted to initiate just after the γ-secretase S3 cleavage site. The resultant protein would be nearly identical to NICD itself, and would be predicted to be highly active and independent of cleavage by γ-secretase (Fig.3b).

We observed substantially higher Notch responsive transcriptional activity in the three cell lines containing Notch fusions, compared with other breast cell lines using a Notch luciferase reporter (Fig.3c). Thus, each of the three Notch fusions is capable of activating the expression of Notch responsive genes. Using an antibody specific to the γ-secretase cleaved active form of the NOTCH1-NICD, both HCC1599 and HCC2218 exhibit high levels of NICD, consistent with the fusion protein acting as a substrate for activation by γ-secretase (Fig.3d). HCC1187, which contains a NOTCH2 fusion gene, exhibits little NOTCH1-NICD. Most breast cancer lines express wild-type NOTCH1 (Fig. 3d, middle panel), however, only the two cell lines with NOTCH1 fusion alleles show
high levels of activated NICD. Each of the three fusion alleles, co-transfected with a Notch reporter plasmid, induced Notch-responsive transcription equivalent to NICD itself (Fig.3e).

The Notch fusions represent two functional classes with respect to dependence on γ-secretase activity. BrCa10040, HCC2218, and HCC1599 fusions are dependent on S3 cleavage for activity and sensitive to γ-secretase inhibitors (GSIs). The HCC1187 fusion class is independent of S3 cleavage. Stable Notch reporter lines were established from each of the three Notch fusion index lines and treated with the γ-secretase inhibitor DAPT 16. A dramatic reduction of Notch reporter activity upon DAPT treatment was seen with HCC1599 and HCC2218 fusion alleles (Fig. 4a). On the other hand, Notch reporter activity is only slightly diminished by DAPT in HCC1187, expressing a γ-secretase independent Notch fusion allele. DAPT treatment also dramatically reduced NICD protein levels in both sensitive cell lines (Fig. 4b). Furthermore, index cell lines exhibit dependence on Notch signaling for proliferation and survival (Fig. 4c). The HCC1599 and HCC2218 cell lines exhibit dramatic reductions in proliferation following DAPT treatment. HCC1187, which expresses a GSI-independent NOTCH2 fusion, shows no reduction in proliferation upon DAPT treatment, as do breast cell lines not expressing Notch fusion alleles.

Treatment with DAPT repressed expression of the Notch targets MYC and CCND1 (Fig. 4d), two genes demonstrated to play a key role in mouse mammary tumorigenesis induced by Notch 17,18, further supporting the possibility GSIs may be useful in treating cancers harboring activated Notch alleles. Consistent with this, treatment with DAPT significantly reduced tumor volume in a xenograft tumor model of HCC1599 (Fig. 4e).

Since the discovery of the TMPRSS2-ERG gene fusion in approximately 50% of prostate cancers, emerging evidence suggests that recurrent gene fusions play a more significant role in common solid tumors than previously appreciated. The MAST and Notch aberrations in breast cancer represent novel classes of rare but functionally recurrent gene fusions with therapeutic implications (similar to the ALK fusions in lung cancer). MAST kinase and Notch gene rearrangements are mutually exclusive aberrations in the samples tested, and together, may represent up to 5–7% of breast cancers. The discovery of functionally recurrent MAST and Notch fusions in a subset of breast carcinomas illuminates a promising path for future research and treatment in breast cancer and illustrates the power of next-generation sequencing as a tool in the development of personalized medicine.

Note: Supplementary information is available on the Nature Medicine website.

Methods

Cell lines and specimen collection

Breast cancer cell lines were purchased from the American Type Culture Collection. This study was approved by the respective Internal Review Boards and breast cancer samples were obtained from the University of Michigan and the Breakthrough Breast Cancer Research Centre, Institute of Cancer Research (London, UK).

Paired end transcriptome sequencing

Total RNA was extracted from normal and cancer breast cell lines and breast tumor tissues and the quality of RNA was assessed with the Agilent Bioanalyzer. Transcriptome libraries from the mRNA fractions were generated following the RNA-SEQ protocol (Illumina. Each sample was sequenced in a single lane with the Illumina Genome Analyzer II (40–80 nucleotide read length) or with the Illumina HiSeq 2000 (100 nucleotide read length). Paired-end transcriptome reads passing filter were mapped to the human reference genome (hg18) and UCSC genes with Illumina ELAND software (Efficient Alignment of Nucleotide Databases). Sequence alignments were subsequently processed to nominate gene fusions using the method described earlier 11,12.

Quantitative RT-PCR and long-range PCR

Quantitative RT-PCR assays using SYBR Green Master Mix (Applied Biosystems) were carried out with the StepOne Real-Time PCR System (Applied Biosystems). Relative mRNA levels of each chimera shown were normalized to the expression of GAPDH. To detect the genomic fusion junction in HCC1187 cells, primers were designed flanking the predicted fusion position and PCR reactions were carried out to amplify the fusion fragments.

Constructs used for over-expression studies

The ZNF700-MAST1 fusion ORF from BrCa00001 was cloned into Gateway pcDNA-DEST40 mammalian expression vector (Invitrogen) using LR Clonase II. A plasmid with C-terminus V5 tag was generated and tested for protein expression following transfection in HEK293 cells. A full-length expression construct of MAST2 with DDK tag was obtained from Origene.

Establishment of stable pools of TERT-HME1 cells

The five MAST fusion alleles were cloned with an amino terminal FLAG epitope tag into the lentiviral vector pCDH510-B (SABiosciences). Lentivirus was produced by cotransfecting each of the MAST plasmids with the ViraPower packaging mix (Invitrogen) into 293T cells using FuGene HD transfection reagent (Roche). Thirty-six hours post-transfection the viral supernatants were harvested, centrifuged, and then filtered through a 0.45 micron Steriflip filter unit (Millipore) TERT-HME1 were infected at an MOI of 20 with polybrene at 8 µg / ml. Forty-eight hours post-infection, the cells were split and placed into puromycin-selective media. Stable pools of TERT-HME1 cells expressing the NOTCH fusion alleles, as well as a control NOTCH1 intracellular domain were generated using the same procedures.

Knockdown assay

For siRNA knockdown experiments, multiple independent MAST2 siRNAs from Thermo were used (J-004633-06, J-004633-07, and J-004633-08). All siRNA transfections were carried out using Oligofectamine reagent (Life Sciences. Similar experiments were performed with multiple custom siRNA sequences targeting the ARID1A-MAST2 fusion (Thermo). Lentiviral particles expressing the MAST2 shRNA (Sigma, TRCN0000001733) were transduced using polybrene, according to the manufacturer’s instructions.

Colony formation assay

MDA-MB-468 cells transduced with scrambled or MAST2 shRNA lentivirus particles were plated and selected using puromycin. After 7–8 days the plates were stained with crystal violet to visualize the number of colonies formed. For quantitation of differential staining, the plates were treated with 10% acetic acid and absorbance was read at 750nm.

Mouse Xenograft Models

Four week-old female SCID C.B17 mice were procured from a breeding colony at University of Michigan, maintained by Dr. Kenneth Pienta. Mice were anesthetized using a cocktail of xylazine (80 mg kg−1 IP) and ketamine (10 mg kg−1 IP) for chemical restraint. MAST2 shRNA or scrambled shRNA knockdown MDA-MB-468 breast cancer cells (4 million) or NOTCH1 fusion allele positive HCC1599 breast cancer cell line (5 million) were resuspended in 100ul of 1X PBS with 20% Matrigel (BD Biosciences) and implanted into right and left abdominal-inguinal mammary fat pads. Ten mice were included in each group. Two weeks after tumor implantation, HCC1599 xenograted mice were treated daily with γ-secretase inhibitor (DAPT) dissolved in 5% ethanol in corn oil (IP). All procedures involving mice were approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan.

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Acknowledgements

The authors thank R. Morey for high throughput sequencing support, T. Barrette for hardware and database management, R. Wang, N. Consul, C. Malla, L. Ma, J. Milton, L. Cai, M. Mei for technical help; K. Suleman and W. Yan for help with cytogenetic analysis. D. Appledorn from Essen Bioscience (Ann Arbor, MI) graciously performed Incucyte analyses. The Aims of this project were defined by the Department of Defense Era of Hope Grant (W81XWH-08-0110) to A.M.C. This project was supported in part by an AACR Stand Up to Cancer (SU2C) award to A.M.C. and J.S.R.F., an R01 CA125577 to C.G.K., the National Functional Genomics Center (W81XWH-11-1-0520) supported by the Department of Defense (A.M.C.), and in part by the National Institutes of Health through the University of Michigan’s Cancer Center Support Grant (5 P30 CA46592). A.M.C. is supported by the NCI Early Detection Research Network (U01 CA111275), the Doris Duke Charitable Foundation Clinical Scientist Award, and the Burroughs Welcome Foundation Award in Clinical Translational Research. R.N., M.B.L., and J.S.R.F are funded in part by Breakthrough Breast Cancer. A.M.C. is an American Cancer Society Research Professor and Taubman Scholar.